Plasmonic polymer nanoparticle carriers

Abstract

The present invention relates to the field of nanoparticles, conjugates thereof, and their use in methods of treating or preventing vascular inflammation. The present invention also relates to methods of delivering an agent to a region of a patient's blood vessel, comprising: a) conjugating an agent to a nanoparticle to produce a conjugate; and b) delivering the conjugate to a region of a blood vessel.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to Australian Provisional Patent Application No. 2019900427, filed on February 11, 2019, the contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of nanoparticles, their conjugates, and their use in methods of treating or preventing vascular inflammation. Background Technology

[0004] In Western societies, coronary atherosclerosis is the leading cause of death and disability. Blockage of the coronary arteries reduces blood flow to the heart muscle, damaging the tissue and ultimately leading to myocardial infarction. However, the long-term performance of medical devices used to treat atherosclerosis, such as drug-eluting stents and angioplasty balloons, is limited by chronic inflammation at the site of injury.

[0005] Long-term success of surgical and vascular interventions is limited by neointimal hyperplasia (NIH). In arteries, NIH refers to thickening of the arterial intima following injury such as angioplasty, stent placement, or surgical repair. NIH is also used to describe thickening of venous and prosthetic bypass grafts, which leads to reduced lumen diameter and flow, and ultimately graft occlusion and thrombosis. NIH affects all forms of vascular grafts, including venous and prosthetic catheters used in coronary and peripheral artery bypass grafts, and arteriovenous fistulas (AVFs) created for hemodialysis access.

[0006] Restenosis is a common side effect of endovascular procedures such as stent placement, balloon angioplasty, or vascular surgery. One of the contributing factors to restenosis is the inflammatory and immune response triggered by endovascular surgery. Recurrence of stenosis is a narrowing of the blood vessel, leading to reduced blood flow.

[0007] Therefore, methods for delivering bioactive agents and drugs such as anti-inflammatory drugs into the blood vessels are still needed. Summary of the Invention

[0008] This invention describes novel therapies for targeting bioactive agents or drugs in areas of blood vessels, such as for modulating inflammation in blood vessels, promoting healing, or treating or preventing disease. In particular, the inventors have identified nanoparticles or nano-P... 3 It can be combined with reagents, such as bioactive agents, drugs and imaging agents, while maintaining their biological activity in vitro and in vivo.

[0009] Therefore, in one aspect, the present invention provides a method for delivering a reagent to a region of a patient's blood vessels, comprising:

[0010] a) Conjugating the reagent with nanoparticles to produce a conjugate; and

[0011] b) Deliver the conjugate to the region of the blood vessel.

[0012] In another aspect, the present invention provides a method for regulating inflammation or promoting healing in a region of a patient's blood vessels, the method comprising:

[0013] a) Conjugating bioactive agents with nanoparticles to create conjugates; and

[0014] b) Deliver the conjugate to the region of the blood vessel.

[0015] In another aspect, the present invention provides a method for retaining a bioactive agent in a region of a patient's blood vessels for at least 14 days, comprising:

[0016] a) conjugating the bioactive agent with nanoparticles to produce a conjugate; and b) delivering the conjugate to the blood vessel.

[0017] In another aspect, the present invention provides a conjugate comprising:

[0018] Nanoparticle polymers with an average diameter of about 1 nm to about 50 nm formed by plasma, wherein the plasma contains at least one monomer selected from olefins, alkynes, cycloalkenes, cycloalkynes or mixtures thereof; or aggregates containing two or more of the nanoparticle polymers, wherein the aggregates have an average diameter of about 5 nm to about 500 nm.

[0019] Bioactive agents selected from the group consisting of: anti-inflammatory cytokines; anti-inflammatory drugs; statins and antiproliferative drugs.

[0020] In another aspect, the present invention provides a conjugate comprising:

[0021] Nanoparticle polymers with an average diameter of about 1 nm to about 50 nm formed by plasma, wherein the plasma contains at least one monomer selected from olefins, alkynes, cycloalkenes, cycloalkynes or mixtures thereof; or aggregates containing two or more of the nanoparticle polymers, wherein the aggregates have an average diameter of about 100 nm to about 200 nm.

[0022] And interleukin-10.

[0023] In another aspect, the present invention provides a conjugate comprising:

[0024] Nanoparticle polymers with an average diameter of about 1 nm to about 50 nm formed by plasma, wherein the plasma contains at least one monomer selected from olefins, alkynes, cycloalkenes, cycloalkynes or mixtures thereof; or aggregates containing two or more of the nanoparticle polymers, wherein the aggregates have an average diameter of about 100 nm to about 200 nm.

[0025] And Siloamus.

[0026] In another aspect, the present invention provides a conjugate comprising:

[0027] Nanoparticle polymers with an average diameter of about 1 nm to about 50 nm formed by plasma, wherein the plasma contains at least one monomer selected from olefins, alkynes, cycloalkenes, cycloalkynes or mixtures thereof; or aggregates containing two or more of the nanoparticle polymers, wherein the aggregates have an average diameter of about 100 nm to about 200 nm.

[0028] And sulindac.

[0029] In another aspect, the present invention provides a method for treating or preventing vascular injury or vascular disease, comprising delivering the conjugate disclosed herein to a region of a vascular vessel in a patient in need.

[0030] In another aspect, the present invention provides the use of the conjugates disclosed herein in the preparation of medicaments for treating or preventing vascular injury or vascular disease in patients in need. Attached Figure Description

[0031] The following figures form part of and are included in this specification to further illustrate certain aspects of the invention. A better understanding of the invention can be achieved by referring to one or more of these figures in conjunction with the detailed description of specific embodiments given herein.

[0032] Figure 1 Evaluation of NP3-conjugated IL-4 (NP3+IL-4) as a therapeutic treatment for cardiovascular disease. The direct delivery of therapeutic cargo to sites of reduced vascularity was investigated. A) Rapid conjugation of therapeutic compounds and increased bioavailability of these compounds when delivered in vivo are possible through the inherent properties of the NP3 platform. B) M1 macrophages, which drive vascular injury / cardiovascular pathology, are pro-inflammatory. Directing the macrophage phenotype to their M2 anti-inflammatory state has the potential to mitigate further progression of vascular injury and promote disease regression. Various cytokines from the interleukin-family promote the phenotype shift from M1 to M2, including IL-4 and IL-10. C) The efficacy of NP3-conjugated interleukins in treating cardiovascular pathology was determined using a rat carotid artery model.

[0033] Figure 2 The loading capacity of IL-4 (blue) and IL-10 (red) on NP3 is shown. IL-4 and IL-10 are labeled and conjugated to NP3 using Cy5 labeling. Fluorescence readings of the Cy5-labeled detergent are quantified to determine how much cargo remains in the solution (unconjugated to NP3) to determine the NP3 loading. A) Total cargo constraint is a function of cargo in the solution. B) Binding efficiency of IL-4 and IL-10 to NP3 as a function of loading. C) Emission spectra of bound IL-4 and IL-10 are used to confirm the binding of IL-4 and IL-10.

[0034] Figure 3 Demonstrates the in vitro polarization of M2 macrophages by NP3+IL-4. A) Scanning electron microscopy (SEM) of RAW 264.76 mouse macrophages shows that, compared with the untreated control and NP3-only, NP3+IL-4 treatment increases spreading (top row) and surface roughness (bottom row), consistent with M2 activation. B) Under confocal microscopy, immunostaining of highly expressed M2 enzymes and arginase-1 (ARG-1) (green) shows that NP3+IL-4 significantly upregulates ARG-1 expression, further demonstrating robust M2 activation compared with the control and NP3-only.

[0035] Figure 4 An in vivo model of vascular injury and NP3 retention was presented. a) Procedural workflow for vascular injury: 1. Microsurgical forceps were inserted into the ligated / dissected rat segment of the carotid artery. 2. The forceps were dilated and rotated 360° to simulate balloon injury with over-dilation / stripping. 3. The forceps were removed, and a small-gauge catheter was inserted through the same incision. 4. An NP3+IL-4 solution was delivered through the catheter and incubated in the dissected vessel for 2 minutes. 5. The incision was sutured to restore blood flow. b) Tracking of IL-4 retention in the dissected vessel using Cy5 markers showed that free IL-4 was washed away from the vessel wall immediately after blood flow was restored. However, when NP3 was combined (NP3+IL-4), IL-4 was significantly retained in the vessel, and this persisted at significant levels after 5 days.

[0036] Figure 5 The study revealed an inhibitory mechanism for neointimal formation. A) Immunostaining (yellow / green) of M2 macrophages in the treated carotid artery segment showed a significant increase in the NP3+IL-10 group compared to ablation, NP3+IL-4, and free IL-10. B) Evaluation of endothelial repair by immunostaining showed that NP3+IL4 and NP3+IL-10 restored complete endothelial integrity 14 days post-injury, a phenomenon not observed with treatment with free IL-10.

[0037] Figure 6 shows an analysis of neointimal formation two weeks after therapeutic NP3 delivery. A) Representative histological photographs showing the extent of neointimal formation in each treatment group. B) Quantification of the percentage of neointimal formation in three segments along the length of the treated carotid artery segment. 'Proximal' and 'distal' markings indicate the orientation of the vascular anastomosis closer to the heart. Stripping injury resulted in approximately 60% vascular occlusion after two weeks. This was significantly reduced to approximately 35% and 20% in the NP3+IL-4 and NP3+IL10 groups, respectively. Free IL-10 and NP3 alone had no significant effect on vascular occlusion, suggesting that the NP3 platform promotes the therapeutic benefit of IL-10.

[0038] Figure 7 This study presents an in vivo analysis of neointimal hyperplasia in a rat carotid artery injury model stained with hematoxylin and eosin (H&E). The compounds tested included the anti-inflammatory cytokine interleukin-10 (IL-10), the anti-proliferative drug sirolimus, and the nonsteroidal anti-inflammatory drug sulindac, delivered freely or conjugated to 200 nm NP3. When delivered to 200 nm NP3, all treatments showed inhibition of neointimal hyperplasia compared to their respective free-delivery controls.

[0039] Figure 8 This study presents an in vivo analysis of vascular reendothelialization in a rat carotid artery injury model stained with von Willebrand factor (VWF). The compounds tested included the anti-inflammatory cytokine interleukin-10 (IL-10), the anti-proliferative drug sirolimus, and the nonsteroidal anti-inflammatory drug sulindac, which were freely delivered or conjugated to NP3 at 200 nm. NP3 delivering IL-10 stimulated vascular healing (endothelialization). Surprisingly, sirolimus delivered by NP3 also stimulated vascular healing.

[0040] Figure 9 This study presents an in vivo analysis of vascular inflammation / macrophage polarization in a rat carotid artery injury model stained with mannose receptor (CD206) and CD68 cell surface receptors. The compounds analyzed included the anti-inflammatory cytokine interleukin-10 (IL-10), which is freely delivered or conjugated to 200 nm NP3, and the nonsteroidal anti-inflammatory drug (sulindac). NP3-IL-10 stimulates an anti-inflammatory response by promoting M2 macrophage polarization.

[0041] Figure 10 The in vivo performance results of IL-10-conjugated 200nm NP3 are shown in a rabbit iliac artery injury model. A) Rabbit iliac artery injury model; B) H&E staining for neointimal hyperplasia; C) CD31 staining for thrombus formation (white dashed line); D) CD68 staining for inflammatory macrophage infiltration (white staining).

[0042] Figure 11 The in vivo performance results of IL-10-conjugated 200nm NP3 in a rabbit iliac artery injury model are shown. A) H&E staining analysis showed that IL-10+NP3-treated vessels had reduced proliferation over seven days compared to untreated controls. B) CD31 staining analysis showed an increased incidence of thrombosis over seven days, which was significantly reduced in IL-10+NP3-treated vessels. C) CD68 staining for vascular inflammation showed that IL-10+NP3 reduced pro-inflammatory macrophage infiltration and inflammation over seven days compared to untreated controls.

[0043] Those skilled in the art will understand that many variations and / or modifications can be made to the above embodiments without departing from the broad general scope of the invention. Therefore, these embodiments are to be considered illustrative rather than restrictive in all respects. Detailed Implementation

[0044] General techniques and definitions

[0045] Unless otherwise specifically defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art (e.g., immunology, molecular biology, immunohistochemistry, biochemistry, oncology, and pharmacology).

[0046] Unless otherwise stated, this invention requires minimal experimentation and utilizes conventional techniques in molecular biology, recombinant DNA technology, immunology, and pharmacology. Such methods are described in, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Fourth Edition (2012), Volume I, Volume II, Volume III; DNA Cloning: A Practical Approach, Vols. I and II (DN Glover, Second Edition., 1995), IRL Press, Oxford, full text; Oligonucleotide Synthesis: A Practical Approach (MJ Gait, ed., 1984), IRL Press, Oxford, full text, especially the papers in these works: Gait, pp. 1-22; Atkinson et al, pp. 35-81; Sproat et al, pp. 83-115; and Wu et al, pp. 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (BD Hames & S.J. Higgins, eds., 1985) IRL Press, Oxford, full text; Immobilized Cells and Enzymes: APractical Approach (1986) IRL Press, Oxford, full text; Perbal, B., A Practical Guide to Molecular Cloning (1984) and Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), the entire series.

[0047] Those skilled in the art will understand that variations and modifications can be made to this invention beyond what has been specifically described. It should be understood that this invention includes all such variations and modifications. This invention also includes, individually or collectively, all steps, features, compositions, and compounds mentioned or indicated in this specification, as well as any and all combinations or any two or more of said steps or features.

[0048] The scope of this invention is not limited to the specific embodiments described herein, which are for illustrative purposes only. As stated herein, functionally equivalent products, compositions, and methods are clearly within the scope of this invention.

[0049] Any particular aspect or embodiment of the invention, or each feature of an embodiment, may be applied by analogy to any other aspect or embodiment of the invention.

[0050] Throughout this specification, unless otherwise specifically stated or required by the context, references to a single step, a composition of substances, a set of steps, or a set of compositions of substances shall be understood to include one or more (i.e., one or more) of those steps, compositions of substances, sets of steps, or sets of compositions of substances.

[0051] As used in this article, the singular forms of “one,” “an,” and “the” include the plural forms of these words unless the context clearly indicates otherwise. For example, mentioning “bacteria” includes multiple types of such bacteria, and mentioning “allergen” refers to one or more allergens.

[0052] In this document, the term “about” covers a 10% tolerance for any value associated with the term. For the avoidance of doubt, it should be understood that the term “about” includes a specific reference to an integer (e.g., “about 10” should be understood to include an explicit reference to 10).

[0053] The term “and / or”, such as “X and / or Y”, should be understood to mean “X and Y” or “X or Y”, and should be understood to provide explicit support for both meanings or whichever meaning.

[0054] Throughout this specification, the word “comprise” or variations thereof such as “comprises” or “comprising” shall be understood to imply inclusion of the elements, integers or steps stated herein, or groups of elements, integers or steps, but not to exclude any other elements, integers or steps, or groups of elements, integers or steps.

[0055] The inventors have demonstrated the production of nanoparticle materials in WO2018 / 112543 (the entire contents of which are incorporated herein by reference), which are described herein as "nanoparticle P". 3 "Nano P3", "Nano P" 3 "Nano P" 3 Materials, NanoP 3 Materials or "NP3". These nano-P 3 The material can serve as a versatile and multifunctional nanocarrier that can be easily functionalized. This is achieved through interaction with nano-P... 3 Diffusion of P within the material to nanoparticles3 Free radical reactions on the material surface and / or through interaction with nano-P 3 Partial / functional group reactions formed on the surface of materials or their conjugates, nano-P 3 The material can be conjugated to a wide range of biomolecules and drugs.

[0056] The inventors have discovered that, for example, delivery of bioactive agents and / or imaging agents conjugated with nanoparticles can prolong the bioavailability and / or durability of agents and drugs delivered in vivo in vascular regions compared to agents that are freely injected into the region of a blood vessel (i.e., delivered in a form not conjugated with the nanoparticles described herein).

[0057] Therefore, in some embodiments, the conjugate is retained in the vascular region for a longer period than the unconjugated bioactive agent is retained in the vascular region.

[0058] This invention provides methods for targeting bioactive agents and modulating inflammation in blood vessels in response to vascular intervention. This invention also provides methods for treating or preventing vascular diseases or injuries.

[0059] Inflammation, restenosis and neointimal hyperplasia

[0060] The methods of the present invention can be used to deliver and / or target reagents, such as bioactive agents or imaging agents, to areas of a patient's blood vessels. Alternatively or additionally, the methods of the present invention can be used to modulate inflammation or promote healing in areas of a patient's blood vessels.

[0061] In one aspect, the present invention provides a method for delivering a reagent to a region of a patient's blood vessels, comprising:

[0062] a) Conjugating the reagent with nanoparticles to produce a conjugate; and

[0063] b) Deliver the conjugate to the region of the blood vessel.

[0064] In one embodiment, the nanoparticles are nanoparticle polymers with an average diameter of about 1 nm to about 50 nm formed by plasma, wherein the plasma contains at least one monomer selected from olefins, alkynes, cycloalkenes, cycloalkynes or mixtures thereof; or aggregates containing two or more of the nanoparticle polymers, wherein the aggregates have an average diameter of about 5 nm to about 500 nm.

[0065] In one implementation, the reagent is a bioactive agent.

[0066] In another embodiment, the reagent is an imaging agent.

[0067] In one aspect, the present invention provides a method for regulating inflammation in a region of a patient's blood vessels, the method comprising:

[0068] a) conjugating a bioactive agent with nanoparticles to produce a conjugate; and b) delivering the conjugate to the region of the blood vessel.

[0069] In one example, inflammation is regulated through macrophage polarization.

[0070] In one aspect, the present invention provides a method for promoting healing in a vascular region of a patient, the method comprising:

[0071] a) conjugating a bioactive agent with nanoparticles to produce a conjugate; and b) delivering the conjugate to the region of the blood vessel.

[0072] Inflammation is triggered when tissue is exposed to one or more types of damage. The response consists of a cascade of events, including the release of various chemical mediators and the recruitment and subsequent activation of circulating blood cells (platelets and leukocytes) to the site of injury.

[0073] Not wanting to be bound by theory, restenosis is considered to be a natural healing process in response to arterial injury that occurs during all types of angioplasty. This highly complex healing process leads to intimal hyperplasia, more specifically, the migration and proliferation of medial smooth muscle cells (SMCs). A problem associated with this arterial healing process is that, in some cases, it does not close. The artery continues to "heal" until it becomes occluded. It should be noted that restenosis is not a re-deposition of plaque-like cholesterol material from the initially occluded artery.

[0074] Unwilling to be bound by theory, it is believed that successful angioplasty of stenotic lesions results in plaque rupture, medial dissection, endothelial cell stripping and destruction, exposure of thrombus-forming collagen, increased release of tissue prothrombin kinase and loss of prostacyclin production, leading to the aggregation of active platelets.

[0075] Activated platelets release several mitogens, including platelet-derived growth factor (PDGF), epidermal growth factor, and transforming growth factor. PDGF possesses mitogenic and chemotactic properties, thus inducing platelet proliferation (endothelial hyperplasia) and reduction of endothelial cells (SMCs) from the mesothelium to the endothelial layer. PDGF induces SMC proliferation by binding to a specific PDGF receptor. Once PDGF binds to its receptor, DNA synthesis occurs, and new cells are replicated. Minor endothelial damage can lead to platelet adhesion and activation, resulting in PDGF release. Therefore, even platelet monolayer deposition is sufficient to induce SMC proliferation.

[0076] Sometimes, deeper arterial injury associated with complex stenotic lesions leads to more extensive platelet deposition and activation, which may result in even greater availability of mitogenic factors. This, in turn, increases SMC proliferation and intimal hyperplasia. Arterial injury induced by angioplasty can lead to the release of PDGF-like compounds not only from platelets but also from macrophages, monocytes, endothelial cells, or the SMCs themselves.

[0077] Activated sclerosing cells (SMCs) from human atherosclerosis or experimental arterial injury secrete PDGF-like molecules, which appear to cause self-sustaining SMC proliferation by releasing their own PDGF-like substances. Therefore, any or all cells that secrete PDGF-related substances (platelets, macrophages, monocytes, endothelial cells, and smooth muscle cells) may contribute to the cascade effect of restenosis after angioplasty.

[0078] One way to prevent restenosis is to stop the proliferation of smooth muscle cells. Therefore, to avoid being bound by theory, some methods to prevent restenosis could be:

[0079] - Reduce platelet adhesion and aggregation at the site of arterial injury;

[0080] - Block the expression of growth factors and their receptors;

[0081] - Develop competitive antagonists for the above-mentioned growth factors;

[0082] - Interfering with receptor signal transduction in responding cells; or

[0083] - Inhibits smooth muscle proliferation.

[0084] Nanoparticles

[0085] The term "nanoparticles" can be related to "nanoparticles" or "nanoparticles". 3 "" or "NP3" are used interchangeably. The terms "nanoparticles" or "nanoparticle P" are used interchangeably. 3 "" or "NP3" refers to nanoparticle materials with a size of less than 100 micrometers, unless otherwise specified or clear from the context of its use. For example, nano-P 3The dimensions can be between approximately 50 to 500, 100 to 500, 200 to 500, 5 to 200, 5 to 100, 5 to 50, 5 to 20, 20 to 100, 100 to 300, or 200 to 400 nm, for example, approximately 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nm, or approximately 5 to approximately 400 nm, or approximately 5 to approximately 300 nm, or approximately 5 to approximately 200 nm, or approximately 5 to approximately 100 nm, or approximately 50 to approximately 100 nm, or approximately 100 to approximately 500 nm. The nanoparticles are in the range of approximately 150 to 500 nm, approximately 180 nm to 500 nm, approximately 100 to 400 nm, approximately 150 to 400 nm, approximately 180 to 400 nm, approximately 100 to 300 nm, approximately 150 to 300 nm, approximately 180 to 300 nm, approximately 100 to 200 nm, approximately 150 to 200 nm, approximately 180 to 200 nm, approximately 150 to 250 nm, approximately 180 to 250 nm, approximately 200 to 400 nm, approximately 200 nm to 300 nm, or a combination thereof. In one embodiment, the nanoparticles are approximately 200 nm. In another embodiment, the nanoparticles are between approximately 50 and 100 nm. It should be understood that the size described herein refers to the diameter or average diameter of the nanoparticle material. The term "nanoparticle P3" is used in this context. 3 The designation “NP3” encompasses both “nanoparticle polymer” and “aggregate” as defined herein, unless otherwise specified or clear from the context of its use. Thus, for example, the dimensions described above also apply to nanoparticles or nanoparticle aggregates. In a preferred embodiment, the nanoparticle material comprises a plasma polymer. The plasma polymer can be formed by the condensation of fragments in plasma, and the material is capable of covalently coupling one or more compounds, such as one or more reagents, including organic or organometallic substances.

[0086] Nanoparticle P3 materials can be homopolymers or copolymers. Examples of suitable nanoparticle P3 materials and methods for deriving suitable nanoparticle P3 materials are described on page 21, line 2 to page 28, line 12 of PCT Publication No. WO2018 / 112543, which is incorporated herein by reference.

[0087] Nanoparticle polymers can be formed in the presence of gases, such as nitrogen, from groups 15, 16, or 17 of the periodic table. Fragments of this gas can be introduced into the nanoparticle polymer. For example, the presence of nitrogen can lead to the formation of nanoparticle polymers or nano-P... 3 The material contains amine, imine, or nitrile groups, or mixtures thereof. Therefore, the nanoparticle polymers disclosed herein may contain nitrogen.

[0088] Nitrogen has been found to be suitable not only as a carrier gas but also as a reactive, non-polymerizable gas. This means that nitrogen can also be incorporated into nanoparticle materials, thereby endowing the resulting functionalized nanoparticles with specific physicochemical properties. Furthermore, nitrogen is also believed to enable different nanoparticle formation modes that would otherwise be impossible without it. It is anticipated that including other gases, such as those in the same nitrogen group, will provide additional degrees of freedom in regulating the nanoparticle formation mechanism and physicochemical properties.

[0089] In one implementation, nano P 3 The material is derived from plasma containing at least one monomer as described herein. Optionally, nano-P 3 The material is formed in the presence of a gas, such as nitrogen, in which fragments of the gas are incorporated into the nanoparticle polymer.

[0090] Nano P 3 The material can have a nitrogen:carbon ratio of about 0.01:1 to about 2:3. For example, the nanoparticle polymer can have a nitrogen:carbon ratio of about 0.05 to about 1, or about 0.1 to about 1, or about 0.15 to about 1, or about 0.2 to about 1, or about 0.25 to about 1, or about 0.3 to about 1, or about 0.35 to about 1, or about 0.4 to about 1, or about 0.45 to about 1, or about 0.5 to about 1, or about 0.55 to about 1, or about 0.6 to about 1, or about 0.65 to about 1. Alternatively, the nanoparticle polymer can have a nitrogen:carbon ratio of about 0.1 to about 1:2. In one example, the nanoparticle polymer can have a nitrogen:carbon ratio of about 0.35 to about 0.5 or about 0.35 to about 1. In another example, the nanoparticle polymer can have a nitrogen:carbon ratio of about 0.38.

[0091] The nano P mentioned in this article 3 The material preferably contains at least one binding site capable of binding one or more compounds (e.g., organic or organometallic compounds, or a second substance as defined herein).

[0092] In one implementation, the nano-P described herein 3 The material contains at least one binding site capable of binding one or more compounds, wherein the binding site contains unpaired electrons capable of binding an organic or organometallic compound or a second substance as defined herein.

[0093] Nano P 3The material may contain unpaired electrons in the polymer. These unpaired electrons may be on or near the surface of the nanoparticles. The unpaired electrons may be located within the nanoparticle material at a depth of 40 nm or less, or within about 30, 20, or 10 nm of the surface, or between about 10 and about 40 nm from the surface, or between about 10 to 30, 20 to 40, or 20 to 30 nm from the surface, or about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 nm from the surface. They can be located at various depths from about 0 to about 40 nm. In some cases, they can be located at depths greater than 40 nm. They can permeate the volume of the nanoparticle material. This allows the material to react with a second substance (such as an organic or organometallic substance) to covalently couple the substance to the nanoparticle polymer and form a conjugate.

[0094] In one implementation, nano-P is provided 3 The material, whose particles have an average diameter of about 5 nm to about 500 nm, the nano-P 3 The material comprises an organic plasma polymer, and the nano-P 3 (Nanoparticle polymers or their aggregates) contain unpaired electrons, thus enabling them to covalently couple with organic or organometallic substances.

[0095] In another embodiment, nanoparticle materials or nano-P 3 The material contains at least one functional part capable of chemically or physically coupling with a second substance.

[0096] Nano P 3 The dimensions of materials can be measured using scanning electron microscopy, transmission electron microscopy, low-angle laser scattering, photon correlation spectroscopy, differential mobility analysis, or some other suitable techniques. Nanoparticles (P) 3 The material particles can have a narrow or wide size distribution. The standard deviation of the particle size distribution can be between about 1% and about 500% of the average particle size, or between about 1 and 200, 1 and 100, 1 and 50, 1 and 20, 1 and 10, 1 and 5, 1 and 2, 10 and 500, 20 and 500, 50 and 500, 100 and 500, 200 and 500, 10 and 100, 10 and 50, or 50 and 100%, for example, about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500%. In some cases, they can be approximately monodisperse, that is, all particles can be approximately the same size (e.g., within about 10%, about 5%, or about 2% of the same diameter).

[0097] Nano P3 Materials may include organic plasma polymers. Plasma polymers are characterized by a heterogeneous, dense, highly cross-linked network. They can be amorphous. These plasma polymers can be generated by the reaction (e.g., ionization and fragmentation) of reactive substances produced in plasma from organic gases and other reactive gases in a gas mixture, or by reactive substances in a plasma / gas mixture generated by the ionization and fragmentation of gases in the gas mixture.

[0098] Nano P 3 Materials can be characterized by a variety of methods, including but not limited to: electron paramagnetic resonance (EPR) spectroscopy, infrared spectroscopy (e.g., Fourier transform infrared spectroscopy), Raman spectroscopy, UV-VIS spectroscopy, elemental analysis (e.g., X-ray photoelectron spectroscopy), soft X-ray spectroscopy, determination of zeta potential, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, gel permeation chromatography, scanning electron microscopy (SEM), transmission electron microscopy (TEM), low-angle laser scattering, photon correlation spectroscopy, differential mobility analysis, elastic recoil detection analysis (ERDA), or neutron scattering.

[0099] Nano P 3 Materials (e.g., nanoparticle polymers or aggregates) can be characterized by one or more of the following features:

[0100] • Broad electron paramagnetic resonance peaks, centered in the range of about 3470 G to about 3520 G, and / or g-factors corresponding to the range of about 2.001 to about 2.005;

[0101] • The spin density, measured by electron paramagnetic resonance, was approximately 10⁻⁶ within approximately 0 to approximately 2 hours after synthesis. 19 To about 10 15 Spin / cm 3 Within the range.

[0102] • The spin density, measured by electron paramagnetic resonance, was approximately 10⁻⁶ within approximately 0 to approximately 240 hours after synthesis. 17 To about 10 15 Spin / cm 3 Within the range.

[0103] • Centered in one or more absorption bands in the infrared spectrum of the following:

[0104] - at approximately 3680-2700cm -1 Within the range;

[0105] - at approximately 1800-1200cm -1 Within the range;

[0106] - at approximately 2330-2020cm-1 Within the range;

[0107] - at approximately 1200-1010cm -1 Within the range; and / or

[0108] - at approximately 1010-700cm -1 Within the range;

[0109] • Centered in one or more absorption bands in the infrared spectrum of the following:

[0110] - at approximately 3600-3100cm -1 Within the range; and / or

[0111] - at approximately 3100-2700cm -1 Within the range;

[0112] • The zeta potential ranges from approximately -100mV to approximately +100mV;

[0113] • The zeta potential measured in solutions with a pH range of approximately 2 to approximately 10 is in the range of approximately -80 mV to approximately +80 mV; or

[0114] The nitrogen:carbon ratio is approximately 0.1:1 to approximately 2:3.

[0115] In one implementation, EPR spectroscopy was used to characterize nano-P 3 Materials, nanoparticle polymers, or aggregates. Nanoparticle polymers or aggregates may exhibit: broad electron paramagnetic resonance peaks centered in the range of about 3470 G to about 3520 G, and / or g-factors corresponding to the range of about 2.001 to about 2.005.

[0116] In one implementation, nano P 3 The spin density of the material, nanoparticle polymer, or aggregate, as measured by electron paramagnetic resonance (EPR), is approximately 10⁻⁶ within approximately 0 to approximately 2 hours after synthesis. 19 To about 10 15 Spin / cm 3 Within the range. After synthesis: approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 minutes, measurements can be taken.

[0117] In one implementation, nano P 3 The spin density of the material, nanoparticle polymer, or aggregate, as measured by electron paramagnetic resonance, is approximately 10 within approximately 0 to approximately 240 hours after synthesis. 17To about 10 15 Spin / cm 3 Within the range. After synthesis: approximately 0.5, approximately 1, approximately 2, approximately 4, approximately 5, approximately 6, approximately 8, approximately 10, approximately 20, approximately 30, approximately 40, approximately 50, approximately 60, approximately 70, approximately 80, approximately 90, approximately 100, approximately 110, approximately 120, approximately 130, approximately 140, approximately 150 hours, approximately 160, approximately 170, approximately 180, approximately 190, approximately 200, approximately 210, approximately 220, approximately 230, or approximately 240 hours, can be measured.

[0118] In another embodiment, infrared spectroscopy (e.g., Fourier transform infrared spectroscopy) is used to characterize nano-P. 3 Materials, nanoparticle polymers, or aggregates. For example, nanoparticle polymers or aggregates may exhibit one or more absorption bands in an infrared spectrum centered on the following:

[0119] · Between approximately 3680 and 2700 cm -1 Within the range;

[0120] · At approximately 1800-1200cm -1 Within the range;

[0121] · Between approximately 2330 and 2020 cm -1 Within the range;

[0122] • Approximately 1200-1010cm -1 Within the range;

[0123] · Between approximately 1010 and 700cm -1 Within the range;

[0124] · At approximately 3600-3100cm -1 Within the range;

[0125] · At approximately 3100-2700cm -1 Within the range; and / or

[0126] • Its mixture.

[0127] In another embodiment, the zeta potential of nanoparticle polymers or aggregates is used to characterize nanoparticle P. 3 Materials, nanoparticles, polymers, or aggregates. For example, nano-P... 3 Materials, nanoparticle polymers, or aggregates may have a zeta potential in the range of about -100 mV to about +100 mV. For example, the zeta potential can be in the range of about -50 mV to about 60 mV.

[0128] In another embodiment, when measured in a solution in a pH range of about 2 to about 10, nano-P3 The zeta potential of the material, nanoparticle polymer, or aggregate is in the range of approximately -80 mV to approximately +80 mV.

[0129] Nano P 3 The materials can exhibit a rough, cauliflower-like surface morphology. This is likely due to their formation through the aggregation of nanoparticle polymers. Therefore, nano-P 3 The material can be an aggregate of nanoparticle polymers. Nanoparticle P 3 Materials and aggregates can be spherical, or typically spherical. The diameter of nanoparticle polymers can range from about 1 to about 50 nm, or from about 5 to 10, 5 to 10, 10 to 50, 20 to 50, or 10 to 30 nm, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nm. Aggregates (and associated nanoparticle polymers) may contain embedded highly reactive free radicals whose amounts decrease over time. Nanoparticle P 3 The material can also embed long-lived and stable free radicals within the delocalized orbitals of carbon clusters. Stable free radicals (secondary free radicals) can be generated by reactions involving highly reactive free radicals (primary free radicals). Nano-P 3 The material can have a hydrophilic surface. This surface can therefore allow for nano-P... 3 Rapid dispersion of the material in water. This allows for the retention of the bioactivity of surface-fixed bioactive molecules. The hydrophilic surface can be the result of free radical oxidation during formation or after formation by exposing the surface to air. After conjugation with one or more second substances, nano-P... 3 Material conjugates can be hydrophilic or hydrophobic. Alternatively, nano-P... 3 Material conjugates can exhibit amphiphilicity.

[0130] Depending on the monomers used or the conditions applied during polymerization, nano-P 3 Materials can be crosslinked. The term "crosslinked" as used herein refers to polymer compositions containing intramolecular and / or intermolecular bonds. These crosslinking bonds can be covalent or non-covalent in nature. Non-covalent bonds include hydrogen bonding, electrostatic bonding, and ionic bonding.

[0131] The potential advantage obtained by crosslinking is the resulting nano-P 3 The stability of the material and the conjugates that may be formed from it. For example, crosslinking can reduce nano-P compared to uncrosslinked similar compositions. 3 The solubility of the material (or the resulting conjugate). Additionally, nano-P 3 The cross-linking properties of materials (or conjugates formed therefrom) can increase the chemical resistance of nanoparticle materials or the resulting conjugates.

[0132] Nano P 3It can be doped with other inorganic elements or compounds, or organometallic compounds, used as image-enhancing contrast agents in medical imaging technology. Nano-P 3 It may be doped with magnetic resonance imaging (MRI) contrast agents, such as iron oxide or gadolinium compounds. Examples of imaging-enhancing contrast agents include: fluorescent dyes (such as Alexa 680, indocyanine green, and Cy5.5); isotopes and radionuclides, such as: 11 C 13 N、 15 O、 18 F, 32 P, 51 Mn, 52m Mn, 52 Fe、 55 Co、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 72 As、 73 Se、 75 Br、 76 Br、 82m Rb、 83 Sr、 86 Y、 90 Y、 89 Zr、 94m Tc, 94 Tc, 99 mTc, 110 In、 111 In、 120 I, 123 I, 124 I, 125 I, 131 I, 154 Gd, 155 Gd, 156 Gd, 157 Gd, 158 Gd, 177 Lu、 186 Re、 188 Re, and 223Ra; paramagnetic ions, such as chromium(III), manganese(II), iron(III), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), or erbium(III); metals, such as lanthanum(III), gold(III), lead(II), and bismuth(III); chromium(III), manganese(II), iron(III) Oxides of iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), or erbium(III); metals such as lanthanum(III), gold(III), lead(II), and bismuth(III), including iron oxide and gadolinium oxide; ultrasound-contrast enhancement agents such as liposomes; and radiopaque agents such as barium, gallium, and thallium compounds. Imaging enhancement contrast agents can be directly incorporated into nano-P 3 In terms of materials, or indirectly incorporated through the use of intermediate functional groups such as chelating agents.

[0133] In an exemplary method, nano-P3 materials can be prepared by plasma polymerization by activating a gas mixture of N2 / C2H2 / Ar at 150 m Torr and applying 50 W of radio frequency power.

[0134] monomer

[0135] The nano P mentioned in this article 3 The material is derived from one or more monomers.

[0136] In one embodiment, one or more monomers are used in gaseous form to form nano-P 3 Material.

[0137] The monomer can be a hydrocarbon. Examples of hydrocarbons include alkenes, alkynes, cycloalkenes, and cycloalkynes.

[0138] Examples of suitable olefin monomers include, but are not limited to: ethylene, propylene, 1-butene, 1-pentene, 1-hexene, 1-heptene, 1-octene, 1-nonene, 1-decene, their isomers or mixtures thereof.

[0139] Examples of suitable alkyne monomers include, but are not limited to: acetylene (calcium carbide gas), propyne, 1-butyne, 1-pentyne, 1-hexyne, 1-heptyne, 1-octyne, 1-nonyne, 1-decyne, their isomers or mixtures thereof.

[0140] Examples of suitable cycloolefin monomers include, but are not limited to: cyclopropylene, cyclobutene, cyclopentene, cyclohexene, cycloheptene, cyclooctene, 1,3-cyclohexadiene, 1,4-cyclohexadiene, 1,5-cyclooctadiene, their isomers or mixtures thereof.

[0141] Examples of suitable cycloalkyne monomers include, but are not limited to: cycloheptyne, cyclooctyne, cyclononyne, their isomers or mixtures thereof.

[0142] In one embodiment, the olefin is used as a monomer. The olefin may be the only monomer used to form the nanoparticle polymer, or it may be used to form a copolymer in the presence of at least one other monomer (e.g., another olefin and / or alkyne, cycloene, or cycloalkyne).

[0143] In one embodiment, acetylene is used as a monomer. The acetylene may be the sole monomer used to form the nanoparticle polymer, or it may be used to form a copolymer in the presence of at least one other monomer (e.g., another acetylene and / or olefin, cycloene, or cycloacetylene). In another embodiment, acetylene is used as a monomer, either alone or in the presence of at least one other monomer.

[0144] In another embodiment, acetylene is used as a monomer in combination with at least one other monomer (e.g., at least one other monomer that is an olefin, alkyne, cycloalkene, or cycloalkyne).

[0145] In one embodiment, the cycloolefin is used as a monomer. The cycloolefin may be the only monomer used to form the nanoparticle polymer, or it may be used to form a copolymer in the presence of at least one other monomer (e.g., another cycloolefin and / or olefin, alkyne, or cycloyne).

[0146] In one embodiment, cycloalkynes are used as monomers. Cycloalkynes may be the sole monomers used to form the nanoparticle polymer, or they may be used to form copolymers in the presence of at least one other monomer (e.g., another cycloalkyne and / or alkene, alkyne, or cycloalkene).

[0147] It can be used to form nano P 3 Other monomers include perfluorinated carbon, ethers, esters, amines, alcohols, or carboxylic acids.

[0148] Examples of suitable perfluorocarbons include, but are not limited to, perfluoroallylbenzene.

[0149] Examples of suitable ethers include, but are not limited to: diethylene glycol vinyl ether, diethylene glycol divinyl ether, diethylene glycol monoallyl ether, or mixtures thereof.

[0150] Examples of suitable amines include, but are not limited to: allylamine, cyclopropylamine, poly(vinylamine), or mixtures thereof.

[0151] Examples of suitable alcohols include, but are not limited to: poly(vinyl alcohol), allyl alcohol, ethanol, or mixtures thereof.

[0152] Examples of suitable carboxylic acids include, but are not limited to, acrylic acid.

[0153] Bioactive agents

[0154] As used herein, the term "bioactive agent" means any agent (e.g., peptide, polypeptide, nucleic acid, or small molecule drug) that has biological and / or pharmacological activity in vivo. Those skilled in the art will understand that bioactive agents or drugs known to prevent restenosis or inflammation, or agents for treating cardiovascular disease, can be suitable agents conjugated with the nanoparticles described herein. Examples of suitable bioactive agents include, but are not limited to, antiplatelet and anticoagulant agents, antithrombotic and fibrinolytic agents, antireplicative and antiproliferative agents, anti-inflammatory drugs, cardiovascular drugs, proteins, peptides, and nucleotides.

[0155] As used herein, the term "peptide" is intended to refer to any polymer comprising amino acids linked by peptide bonds. The term "peptide" is intended to include polymers assembled using ribosomes, as well as polymers assembled by enzymes (i.e., non-ribosomal peptides) and synthetically assembled polymers. In various embodiments, the term "peptide" may be considered synonymous with "protein" or "polypeptide." In various embodiments, the term "peptide" may be limited to polymers containing more than 50 amino acids, or 50 or fewer amino acids. In various embodiments, the term "peptide" is intended to include only amino acids as monomeric units of the polymer, while in various embodiments, the term "peptide" includes additional components and / or modifications to the amino acid backbone. For example, in various embodiments, the term "peptide" may be applied to amino acid core polymers and derivatives of core polymers, such as core polymers having side polyethylene glycol groups or core polymers having amide groups at the amino or carboxyl ends of the amino acid chains.

[0156] "Peptide mimics" can be molecules such as peptides, modified peptides, or any other molecules that biologically mimic the active ligands of biomolecules, such as enzyme substrates or cytokines. For example, peptide mimics can antagonize, stimulate, or modulate the physiological activity of cytokines involved in inflammatory processes. Alternatively, peptide mimics can mimic the activity of their natural proteins in the treatment of cardiovascular diseases.

[0157] Here, the term "protein" refers to an amino acid sequence whose chain length is sufficient to produce higher levels of tertiary and / or quaternary structures. Proteins can have a molecular weight in the range of about 300 Da to about 150 kDa. Proteins can have a molecular weight greater than 150 kDa or less than 300 Da.

[0158] Antiplatelet and anticoagulant agents

[0159] Antiplatelet agents and anticoagulants prevent adhesion and platelet aggregation. In one example, the bioactive agent is an antiplatelet agent. In another example, the bioactive agent is an anticoagulant.

[0160] Examples of suitable antiplatelet agents include, but are not limited to, aspirin and dipyridamole. Aspirin is classified as an analgesic, antipyretic, anti-inflammatory, and antiplatelet drug. It has been clinically tested and demonstrated to reduce the risk of sudden death and / or non-fatal re-infarction in patients following myocardial infarction (heart attack). The proposed mechanism by which aspirin works directly involves platelets. It blocks platelets in a certain way, restricting clotting. This prevents the cascade of platelet aggregation and subsequent restenosis found in thrombi. Therefore, aspirin is a potential restenosis inhibitor. Dipyridamole is a drug similar to aspirin because of its antiplatelet properties. Dipyridamole is also classified as a coronary vasodilator. It increases coronary blood flow through the initial selective dilation of the coronary arteries without altering systemic blood pressure or blood flow in peripheral arteries. These vasodilatory properties are thought to be potentially beneficial in the prevention of restenosis.

[0161] Examples of suitable anticoagulant drugs include, but are not limited to, heparin, coumarin, protamine, and hirudin. These drugs act as anticoagulants by preventing the production of thrombin (a binding agent that causes blood clotting). This can also reduce the cascade of platelet aggregation at the lesion site, potentially reducing restenosis. The use of protamine in the presence of heparin results in protamine acting as a heparin antagonist, blocking the effect of heparin. However, protamine can be used alone as an anticoagulant. Hirudin was chosen because it is not normally present in humans. Hirudin is a drug found in the salivary glands of leeches. It is a highly concentrated anticoagulant that works in a similar manner to heparin, coumarin, and protamine.

[0162] Antithrombotic and fibrinolytic agents

[0163] In one example, the bioactive agent is an antithrombotic agent. In another example, the bioactive agent is a plasminogen activator.

[0164] Examples of antithrombotic agents and fibrinolytic agents include, but are not limited to, glycoprotein IIb / IIIa inhibitors, direct thrombin inhibitors, heparin, low molecular weight heparin, platelet adenosine diphosphate (ADP) receptor inhibitors, plasminogen activators (including streptokinase, urokinase, recombinant tissue plasminogen activator, reteplase, and tenecteplase), enzymes (including streptokinase, urokinase, tissue plasminogen activator (tPA), and plasmin), or mixtures thereof.

[0165] Anti-replication and anti-proliferation agents

[0166] Several types of drugs can interrupt cell replication. Without being bound by theory, antimitotic agents (cytotoxic agents) work directly to prevent cell mitosis (replication), while antimetabolites prevent deoxyribonucleic acid (DNA) synthesis, thus preventing replication. In one example, the bioactive agent is an antireplication drug. In another example, the bioactive agent is an antiproliferative agent.

[0167] Examples of suitable anti-replication drugs include, but are not limited to, methotrexate, colchicine, azathioprine, vincristine, vinblastine, fluorouracil, adriamycin, and mutamycin.

[0168] Examples of suitable antiproliferative agents include, but are not limited to, mTOR inhibitors (including sirolimus, everolimus, and ABT-578); paclitaxel; and antitumor agents (including alkylating agents such as cyclophosphamide, nitrogen mustard, chlorambucil, melphalan, carmustine, lomustine, ifosfamide, procarbazine, dacarbazine, temozolomide, altretamine, cisplatin, carboplatin, and oxaliplatin). In one embodiment, the bioactive agent is paclitaxel.

[0169] In another embodiment, the bioactive agent is sirolimus ((1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-12-[(2R)-1-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]propyl-2-yl]-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.1.0]). 4,9Sirolimus (CAS No. 53123-88-9) is a macrolide compound believed to inhibit T-cell and B-cell activation by reducing the sensitivity of T-cells and B-cells to IL-2 through mTOR inhibition. Sirolimus is an immunosuppressive drug and has previously been reported as a contraindication to wound healing. Impaired wound healing at surgical sites is commonly observed in patients receiving this drug. Vascular areas treated with sirolimus are known to experience delayed reendothelialization due to the drug's antiproliferative effect. Sirolimus is shown in Formula I.

[0170] Formula I:

[0171] - Anti-inflammatory agents

[0172] Anti-inflammatory bioactive agents or drugs can also be used to locally suppress inflammation caused by damage to the cavitary tissue during angioplasty.

[0173] In one example, the bioactive agent is an anti-inflammatory agent. For instance, an anti-inflammatory drug can be an anti-inflammatory drug or a biomolecule.

[0174] Suitable anti-inflammatory agents include, but are not limited to, corticosteroids such as dexamethasone, betamethasone, and prednisone, and broad-spectrum immunosuppressants such as sulindac (2-[(3Z)-6-fluoro-2-methyl-3-[(4-methylsulfinylphenyl)methylene]ind-1-yl]acetic acid; CAS No. 38194-50-2), naproxen (CAS No. 22204-53-1), and aspirin (CAS No. 50-78-2). In one embodiment, the bioactive agent is sulindac (2-[(3Z)-6-fluoro-2-methyl-3-[(4-methylsulfinylphenyl)methylene]ind-1-yl]acetic acid; CAS No. 38194-50-2). Sulindac, represented by Formula II, is a nonsteroidal anti-inflammatory drug.

[0175] Formula II:

[0176]

[0177] Suitable anti-inflammatory agents include cytokines or fragments thereof. Cytokines are small proteins (~5-20 kDa) that are important in cell signaling. Inflammation is characterized by the interaction between pro- and inflammatory cytokines. Cytokines can include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factor. Suitable examples include, but are not limited to, anti-inflammatory cytokines such as IL-4, IL-10, IL-13, IFN-α, and transforming growth factor-β.

[0178] It is known in the art that macrophage polarization can also be activated by interleukins. The M1 phenotype is pro-inflammatory, and the M2 phenotype is anti-inflammatory. Therefore, those skilled in the art will understand that interleukins that activate the M2 anti-inflammatory phenotype are known to be suitable bioactive agents conjugated to nanoparticles as described herein. In one example, the bioactive agent is a cytokine or a fragment thereof. In one example, the bioactive agent is interleukin-4 (IL-4). In another example, the bioactive agent is interleukin-10 (IL-10).

[0179] Cardiovascular reagents

[0180] In one example, the bioactive agent can be a drug used to treat cardiovascular disease. Suitable drugs include, but are not limited to, ACE inhibitors, antihypertensive protein receptor blockers, calcium channel blockers, vasodilators, and statins (also known as HMG-CoA reductase inhibitors). For example, the bioactive agent is a statin such as simvastatin, pitavastatin, lovastatin, fluvastatin; or a mixture thereof. In one embodiment, the bioactive agent is simvastatin.

[0181] It is also anticipated that nitric oxide releasers can be suitable agents for conjugation with nanoparticles as described herein.

[0182] Antibody

[0183] In one embodiment, the bioactive agent is an antibody. The antibody may be one capable of targeting the bioactive agent to the correct location in the blood vessel. Alternatively or optionally, the antibody may be an antagonist, such as a cytokine inhibitor.

[0184] As used herein, the term "antibody" should be understood to encompass proteins containing a variable region consisting of one or more immunoglobulin chains, such as those containing V. L peptides and containing V H The polypeptide. Antibodies may also contain constant domains, some of which can be arranged into crystallizable (Fc) constant regions or constant fragments or segments. V H and VL The interaction forms an Fv, which contains an antigen-binding region capable of specifically binding to one or more closely related antigens. Typically, the light chain from mammals is a κ or λ light chain, and the heavy chain from mammals is an α, δ, ε, γ, or μ chain. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY antibodies), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The term “antibody” also includes humanized antibodies, deimmunized antibodies, non-depleted antibodies, inactive antibodies, primate-derived antibodies, human antibodies, and chimeric antibodies. As used herein, the term “antibody” is also intended to include forms other than full-length, complete, or intact antibody molecules, such as Fab, F(ab')2, and Fv capable of binding epitope determinants. These forms may be referred to as antibody “fragments.” These antibody forms retain some ability to selectively bind target proteins, examples of which include, but are not limited to, the following:

[0185] (1)Fab, a fragment containing a monovalently bound segment of an antibody molecule, which can be digested with papain to produce a complete light chain and a portion of a heavy chain.

[0186] (2) Fab', a fragment of the antibody molecule, can be obtained by treating the whole antibody with pepsin and then reducing it to produce the complete light chain and a portion of the heavy chain; each antibody molecule yields two Fab' fragments;

[0187] (3)(Fab')2, a fragment of antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab)2 is a dimer of two Fab' fragments linked together by two disulfide bonds;

[0188] (4)Fv is defined as a genetically engineered segment containing both light chain variable regions and heavy chain variable regions, and is represented by two strands;

[0189] (5) Single-chain antibodies (“SCAs”) are defined as genetically engineered molecules containing variable regions of both light and heavy chains, linked by suitable polypeptide linkers to form a single-chain molecule for genetic fusion; such single-chain antibodies may be in the form of multimers, such as biantibodies, triantibodies, and tetraantibodies, and may or may not be multispecific; and

[0190] (6) Single-domain antibodies are usually variable heavy chain domains that lack light chains.

[0191] Therefore, the antibodies described herein may include isolated heavy chains, light chains, Fab, Fab', F(ab')2, Fc, variable light chain domains lacking any heavy chain, variable heavy chain domains lacking any light chain, and Fv. Such fragments can be generated by recombinant DNA technology or by enzymatic or chemical separation of intact immunoglobulins. Any antibody or fragment thereof described herein and known in the art can be used with the nanoparticles disclosed herein. 3 Particle fusion.

[0192] Therefore, bioactive agents can be antibodies, proteins, or peptides that target factors involved in the inflammatory process.

[0193] In this article, the term "targeting ligand" refers to a molecule that binds to or interacts with a target molecule. Typically, the nature of the interaction or binding is non-covalent, such as through hydrogen bonds, electrostatics, or van der Waals forces; however, binding can also be covalent.

[0194] As used in this article, the term "ligand" refers to a compound that targets a biomarker. Examples of ligands include, but are not limited to, proteins, peptides, antibodies, antibody fragments, sugars, carbohydrates, glycans, cytokines, chemokines, nucleotides, lectins, lipids, receptors, steroids, neurotransmitters, cluster nomenclature / differentiation (CD) markers, imprinted polymers, etc.

[0195] Examples of targeting ligands include, but are not limited to, nuclear localization signals (e.g., KR[PAATKKAGQA]KKKK), RGD, NGR, folic acid, transferrin, GM-CSF, galactosamine, anti-VEGFR, anti-ERBB2, anti-CD20, anti-CD22, anti-CD19, anti-CD33, anti-CD25, anti-tenosin, anti-CEA, anti-MUC1, anti-TAG72, anti-HLA-DR10, or mixtures thereof.

[0196] Polynucleotides

[0197] Bioactive agents can be polynucleotides capable of modulating inflammatory processes. As used herein, the term "polynucleotide" should be understood to encompass DNA, RNA, antisense polynucleotides, ribozymes, interfering RNA, siRNA, microRNA, and any other polynucleotides known in the art. Polynucleotides can encode proteins or functional RNAs (such as interfering RNA) capable of disrupting inflammation. Therefore, polynucleotides can be polynucleotide carriers or plasmids.

[0198] Examples of gene targeting agents include, but are not limited to, DNA (gDNA, cDNA), RNA (sense RNA, antisense RNA, mRNA, tRNA, rRNA, small interfering RNA (siRNA), short hairpin RNA (ShRNA), microRNA (miRNA), small nucleolar RNA (SnoRNA, small nucleus (snRNA)), ribozymes, aptamers, DNases, antisense oligonucleotides, vectors, plasmids, other ribonuclease-type complexes, and mixtures thereof. For example, a bioactive agent could be a siRNA targeting the p65 subunit of NF-κB, thereby modifying NF-κB-mediated inflammation or a gene targeting IkB kinase or AP-1 encoding cytokine expression and / or cytokine receptor expression.

[0199] stem cells

[0200] Bioactive agents can be stem cells, such as skeletal muscle myoblasts, bone marrow-derived stem cells, bone marrow-derived monocytes, bone marrow-derived hematopoietic stem cells and endothelial progenitor cells, mesenchymal matrix / stem cells, cardiac stem cells and progenitor cells, induced pluripotent stem cells, or mixtures thereof. For example, stem cells can be mesenchymal matrix / stem cells that have adopted an immunosuppressive phenotype in the presence of pro-inflammatory cytokines.

[0201] Imaging agent

[0202] In one embodiment, the reagent is an imaging agent. The imaging agent can be used in vivo to study vascular structure, function, and angiogenesis. Examples of suitable imaging agents include, but are not limited to, luciferase; fluorescently labeled dyes and antibodies; contrast agents (including iopamidol, iohexol, and iodocyanine); barium sulfate; indocyanine green (ICG), and mixtures thereof.

[0203] Imaging agents can be image-enhancing contrast agents. Examples of suitable image-enhancing contrast agents include, but are not limited to, fluorescent dyes (e.g., Alexa 680, indocyanine green, and Cy5.5); isotopes and radionuclides, such as: 11 C 13 N、 15 O、 18 F, 32 P, 51 Mn, 52m Mn, 52 Fe、 55 Co、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 72 As、 73 Se、 75 Br、 76Br、 82m Rb、 83 Sr、 86 Y、 90 Y、 89 Zr、 94m Tc, 94 Tc, 99 mTc, 110 In、 111 In、 120 I, 123 I, 124 I, 125 I, 131 I, 154 Gd, 155 Gd, 156 Gd, 157 Gd, 158 Gd, 177 Lu、 186 Re、 188 Re, and 223 Ra; paramagnetic ions, such as chromium(III), manganese(II), iron(III), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), or erbium(III); metals, such as lanthanum(III), gold(III), lead(II), and bismuth(III); chromium(III), manganese(II), iron(III) Oxides of iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), or erbium(III); metals such as lanthanum(III), gold(III), lead(II), and bismuth(III), including iron oxide and gadolinium oxide; ultrasound-contrast enhancement agents such as liposomes; and radiopaque agents such as barium, gallium, and thallium compounds. Imaging enhancement contrast agents can be directly conjugated to nano-P 3 In terms of materials, or indirectly by using intermediate functional groups (such as chelating agents) to bind to nano-P 3 In terms of materials.

[0204] Conjugate

[0205] In this document, the term "conjugate" refers to a molecule formed by attaching one or more compounds to a polymer of nanoparticles or an aggregate comprising polymer of nanoparticles. "One or more compounds" can be a bioactive agent as defined herein. The connection can be via covalent bonds or electrostatic interactions.

[0206] The nanoparticle polymers, aggregates, or nanoparticles described in this article 3Material and reagent conjugation. The reagent can be a bioactive agent or an imaging agent. In one embodiment, the reagent is a bioactive agent. In another embodiment, the reagent is an imaging agent. The binding of the reagent can be adjusted by changing the pH of the reaction conditions during the conjugation process. The pH of the solution is adjusted by protonation or deprotonation of surface functional groups (such as amine and carboxylic acid groups) of nano-P 3 The charge. For example, positively charged conjugates with nano-P 3 The bonding of materials can be achieved by increasing the content of nano-P 3 The pH of the solution of the materials and reagents is adjusted to improve the pH. In highly alkaline media, nano-P... 3 By deprotonating the carboxyl surface groups to become negatively charged, the repulsion between negatively charged particles also stabilizes the nanoparticles. Alternatively, negatively charged conjugates can interact with nano-P... 3 The bonding of materials can be achieved by reducing the content of nano-P 3 The pH of the solution of materials and reagents is adjusted to improve the pH.

[0207] This article discloses nano P 3 Use of materials (e.g., nanoparticle polymers, aggregates or mixtures thereof) in the formation of conjugates.

[0208] Conjugates may contain only a single reagent. Alternatively, conjugates may contain two or more different reagents, such as two, three, or four second reagents.

[0209] Nano P 3 Materials can typically be directly coupled to reagents, such as bioactive agents or imaging agents, for example, via covalent or ionic coupling. When the nanoparticle polymer or aggregate is a plasmon polymer, the coupling process is usually rapid and can be carried out under mild conditions. Coupling can be achieved by means of unpaired electrons (i.e., radical sites) in the polymer structure or by means of nanoparticles. 3 Functional groups generated on the material can be achieved either by adding suitable bioactive agents or by reacting with air (or another gas) or nano-P. 3 The material is exposed to some other fluid reactions and binds to the resulting conjugate. Nano P 3 The material may include monomeric units, which include functional portions capable of chemically coupling reagents. One or more reagents are combined with nano-P... 3 The conjugation of materials can introduce functional groups onto or into the resulting conjugates, which can be used for further chemical reactions or as binding sites for processes such as biochemical / biological processes that occur under in vitro or in vivo conditions.

[0210] In this article, the functional part (or "functional group") refers to the functional group existing in monomers and nanoparticles. 3 Materials or materials containing nano-P 3The atomic groups on the conjugate of the material can react with other complementary functional groups, such as those present on the reagent. Functional groups include, but are not limited to, the following: carboxylic acids (-(C=O)OH), carbonyl groups, primary or secondary amines (-NH2, -NH-), nitric oxide, maleimide, thiols (-SH), sulfonic acids (-(O=S=O)OH), carbonates, carbamates (-O(C=O)N<), hydroxyl groups (-OH), aldehydes (-(C=O)H), ketones (-(C=O)-), hydrazines (>NN<), isocyanates, isothiocyanates, phosphoric acids (-O(P=O)OHOH), phosphonic acids (-O(P=O)OHH), haloacetyl groups, haloalkanes, acryloyl groups, aryl fluorides, hydroxylamine, disulfides, vinyl sulfones, vinyl ketones, diazonides, ethylene oxide, and aziridine, or mixtures thereof.

[0211] Therefore, in one aspect, the present invention provides a conjugate comprising:

[0212] Nanoparticle polymers with an average diameter of about 1 nm to about 50 nm formed by plasma, wherein the plasma contains at least one monomer selected from olefins, alkynes, cycloalkenes, cycloalkynes or mixtures thereof; or aggregates containing two or more of the nanoparticle polymers, wherein the aggregates have an average diameter of about 5 nm to about 500 nm.

[0213] Bioactive agents selected from the group consisting of: anti-inflammatory cytokines; anti-inflammatory drugs; statins and antiproliferative drugs.

[0214] In one embodiment, the average diameter of the aggregate ranges from about 5 to about 500 nm, or about 5 to about 400 nm, or about 5 to about 300 nm, or about 5 to about 200 nm, or about 5 to about 100 nm, or about 50 to about 100 nm, or about 100 to about 500 nm, or about 150 to about 500 nm, or about 180 nm to about 500 nm, or about 100 to about 400 nm, or about 150 to about 400 nm, or about 180 to about 400 nm, or about 100 to about 300 nm, or about 150 to 300 nm, or about 180 to 300 nm, or about 100 to about 200 nm, or about 150 to about 200 nm, or about 180 to about 200 nm, or about 150 to about 250 nm, or about 200 to about 400 nm, or about 200 nm to about 300 nm, or a mixture thereof. In one embodiment, the aggregate has an average diameter of about 200 nm. In another embodiment, the aggregate has an average diameter of about 100 nm. In one embodiment, the average diameter of the aggregate ranges from about 100 nm to about 200 nm. In yet another embodiment, the average diameter of the aggregate ranges from about 50 nm to about 100 nm.

[0215] In one embodiment, the anti-inflammatory cytokine is IL-4. IL-4 may be administered at doses of approximately 0.5 μg, approximately 0.6 μg, approximately 0.7 μg, approximately 0.8 μg, approximately 0.9 μg, approximately 1.0 μg, or approximately 1.1 μg. 9 Nano P 3 or equivalent concentration of nano P in the amount of substitution 3 Loading amount and nano P 3 Combine.

[0216] In one embodiment, the anti-inflammatory cytokine is IL-10. IL-10 may be administered in doses of approximately 0.5 μg, approximately 0.6 μg, approximately 0.7 μg, approximately 0.8 μg, approximately 0.9 μg, approximately 1 μg, approximately 1.1 μg, approximately 1.2 μg, approximately 1.3 μg, or approximately 1.4 μg IL-10 / 10 9 Nano P3 or equivalent concentration of nano P3 by substitution 3 The loading amount is combined with nano-P3. In one example, IL10 can be loaded at approximately 1.3 μg IL-10 / 10 9 The loading amount of nano-P3 or the equivalent concentration of nano-P3 of the substitution amount is combined with nano-P3.

[0217] Therefore, in another aspect, the present invention provides a conjugate comprising:

[0218] Nanoparticle polymers with an average diameter of about 1 nm to about 50 nm formed by plasma, wherein the plasma contains at least one monomer selected from olefins, alkynes, cycloalkenes, cycloalkynes or mixtures thereof; or aggregates containing two or more of the nanoparticle polymers, wherein the aggregates have an average diameter of about 100 nm to about 200 nm.

[0219] And IL-10. As described herein, alternative average diameters may be used.

[0220] In one implementation, the antiproliferative drug is sirolimus. Sirolimus can be administered in doses of about 1.5 μg to about 3 μg, or about 1.5 μg to about 2.5 μg, or about 2 μg to about 2.5 μg, or about 2 μg to about 3 μg sirolimus / 10 9 Nano P 3 or equivalent concentration of nano P in the amount of substitution 3 Loading amount and nano P 3 Combination. In one example, sirolimus can be approximately 2.50 μg sirolimus / 10 9 Nano P 3 or equivalent concentration of nano P in the amount of substitution 3 Loading amount and nano P 3 Combine.

[0221] The inventors were surprised to discover that, with nano-P 3 Bound sirolimus increases reendothelialization, thereby promoting vascular healing. This finding is contrary to the effect of free sirolimus.

[0222] Therefore, in another aspect, the present invention provides a conjugate comprising:

[0223] Nanoparticle polymers with an average diameter of about 1 nm to about 50 nm formed by plasma, wherein the plasma contains at least one monomer selected from olefins, alkynes, cycloalkenes, cycloalkynes or mixtures thereof; or aggregates containing two or more of the nanoparticle polymers, wherein the aggregates have an average diameter of about 100 nm to about 200 nm.

[0224] And sirolimus. As described in this article, an alternative average diameter may be used.

[0225] In one embodiment, the anti-inflammatory drug is sulindac. Sulindac can be administered in doses of about 2 μg to about 3.5 μg, or about 2.5 μg to about 3.5 μg, or about 2.5 μg to about 3.1 μg sulindac / 10 9 Nano P 3 or equivalent concentration of nano P in the amount of substitution 3 Loading amount and nano P 3 In combination. In one example, sulindac can be administered at approximately 3.05 μg sulindac / 10 9 Nano P 3 or equivalent concentration of nano P in the amount of substitution 3 Loading amount and nano P 3 Combine.

[0226] Therefore, in another aspect, the present invention provides a conjugate comprising:

[0227] Nanoparticle polymers with an average diameter of about 1 nm to about 50 nm formed by plasma, wherein the plasma contains at least one monomer selected from olefins, alkynes, cycloalkenes, cycloalkynes or mixtures thereof; or aggregates containing two or more of the nanoparticle polymers, wherein the aggregates have an average diameter of about 100 nm to about 200 nm.

[0228] And sulindac. As described in this article, an alternative average diameter may be used.

[0229] Nanoparticle polymers, aggregates, or conjugates thereof are expected to be incorporated into suitable scaffolds for treating incisions / trauma or vascular injuries. For example, nanoparticle polymers, aggregates of their conjugates, can be used on or in implants such as cardiac patches, vascular grafts, and scaffolds.

[0230] Preparation of conjugates

[0231] Nano P 3 Materials and Nanoparticles 3 The production details of the conjugate are described on page 49, line 18 to page 71, line 7 of PCT Publication No. WO2018 / 112543, which is incorporated herein by reference. Exemplary methods for producing the conjugate are provided below.

[0232] Nanoparticles of P3 can be synthesized via plasma polymerization by activating a gas mixture of N2 / C2H2 / Ar at 150 mTorr and applying a radio frequency power of 50 W. The loading capacity and binding efficiency of nanoparticles of P3 for bioactive agents such as IL-4 and IL-10 can be measured using fluorescently labeled molecules such as Cy5 to establish optimal incubation parameters for in vitro and in vivo applications.

[0233] The bioactive agent can be mixed with nano-P3 in ultrapure water at a total reaction volume of 1 ml and incubated at room temperature for 1 hour. After 1 hour of incubation, the residue can be washed and the binding kinetics can be determined on a Clariostar monochromator microplate reader (BMG Laboratory Technology, Germany).

[0234] Pharmaceutical Composition

[0235] Conjugates may be present in pharmaceutical compositions. Details of suitable pharmaceutical compositions and methods for obtaining suitable pharmaceutical compositions are described on page 40, line 7 to page 44, line 26 of PCT Publication no. WO2018 / 112543, which is incorporated herein by reference.

[0236] Delivery of conjugates to blood vessels

[0237] This article discloses methods for targeting bioactive agents to regions of blood vessels, methods for modulating inflammation in regions of blood vessels, and methods for retaining bioactive agents at delivery sites in blood vessels by conjugating bioactive agents with nanoparticles to create conjugates and delivering the conjugates to regions of blood vessels.

[0238] The conjugate can be delivered to a blood vessel by any suitable delivery method known in the art. Examples of suitable delivery methods include, but are not limited to, catheters, stents, stent grafts, grafts and valves, or direct injection into the blood vessel.

[0239] In one example, the catheter is an occlusive perfusion catheter. In another example, the catheter is a dispensing balloon catheter.

[0240] Following delivery, the conjugate remains at the delivery site in the blood vessel. In one embodiment, the conjugate remains in the blood vessel region for a longer period than the unconjugated bioactive agent. For example, the conjugate remains in the blood vessel region for at least 1.2, 1.2, 1.5, 1.7, or at least twice the time the unconjugated bioactive agent remains, or the conjugate may remain at the delivery site for at least 1 time, or at least 2 days, or at least 3 days, or at least 4 days, or at least 5 days, or at least 6 days, or at least 7 days, or at least 8 days, or at least 9 days, or at least 10 days, or at least 11 days, or at least 12 days, or at least 13 days, or at least 14 days. In one example, the conjugate remains at the delivery site for at least 1 day. In another example, the conjugate remains at the delivery site for at least 5 days. In yet another example, the conjugate remains at the delivery site for at least 14 days.

[0241] processing method

[0242] This document also discloses methods for treating or preventing vascular injury or vascular disease, methods comprising the steps of delivering to a subject a conjugate as defined herein or a pharmaceutical composition comprising a conjugate as defined herein. The term “treatment” is used herein to encompass both therapeutic and preventative treatment. Therefore, the treatment methods disclosed herein may encompass methods for preventing one or more symptoms of a disease, condition, or illness. It should be understood that “treatment” can be interpreted as achieving relief of any one or more symptoms of a disease, condition, or illness. Therefore, “treatment” includes a reduction in vascular occlusion or neointimal formation and / or an increase in the rate of reendothelialization relative to a patient who has not received a conjugate disclosed herein or who has only received an unconjugated bioactive agent. As used herein, “promoting healing” means an increase in any indicative marker of healing relative to a patient who has not received a conjugate disclosed herein or who has only received an unconjugated bioactive agent. “Healing” may refer to the healing of a wound in a blood vessel. It should be understood that the term “healing” includes processes such as endothelialization. Therefore, any reference to promoting healing should be understood as promoting endothelialization.

[0243] In one aspect, the present invention provides a method for treating or preventing vascular injury or vascular disease, comprising delivering a conjugate, as defined herein, to a region of a vascular vessel in a patient in need.

[0244] Therefore, in one example, the present invention provides a method for treating or preventing vascular injury or vascular disease, the method comprising delivering a conjugate comprising:

[0245] Nanoparticle polymers with an average diameter of about 1 nm to about 50 nm formed by plasma, wherein the plasma comprises at least one monomer selected from olefins, alkynes, cycloalkenes, cycloalkynes, or mixtures thereof; or aggregates comprising two or more of the nanoparticle polymers, wherein the aggregates have an average diameter of about 100 nm to about 200 nm; and

[0246] i) Interleukin-10; or

[0247] ii) Sulindac; or

[0248] iii) Sirolimus.

[0249] In another aspect, the present invention provides a method for promoting healing, the method comprising delivering a conjugate, as defined herein, to a region of a blood vessel in a patient in need.

[0250] Therefore, in one example, the present invention provides a method for promoting healing, the method comprising delivering a conjugate comprising:

[0251] Nanoparticle polymers with an average diameter of about 1 nm to about 50 nm formed by plasma, wherein the plasma comprises at least one monomer selected from olefins, alkynes, cycloalkenes, cycloalkynes, or mixtures thereof; or aggregates comprising two or more of the nanoparticle polymers, wherein the aggregates have an average diameter of about 100 nm to about 200 nm; and

[0252] i) Interleukin-10; or

[0253] ii) Siromoos

[0254] This article also discloses the use of the conjugates defined herein in the preparation of medicaments for the treatment or prevention of a patient’s condition.

[0255] In one aspect, the present invention provides the use of the conjugates as defined herein in the preparation of medicaments for treating or preventing vascular injury or vascular disease in patients in need.

[0256] In one aspect, the present invention provides the use of the conjugates as defined herein in the preparation of medicaments for promoting healing in patients in need.

[0257] In one implementation, the conjugates defined herein are used as a drug or for the preparation of a drug.

[0258] In another implementation, the conjugate, as defined herein, is used to treat or prevent vascular diseases.

[0259] In another implementation, the conjugate, as defined herein, is used to promote healing.

[0260] In another aspect, the present invention provides conjugates as defined herein for use in the treatment or prevention of vascular diseases.

[0261] In another aspect, the present invention provides conjugates as defined herein when used to promote healing.

[0262] For example, when a suitable compound is added to a pharmaceutical composition, the conjugate may be provided in an "effective amount". The phrase "effective amount" refers to the amount of conjugate that will elicit the desired biological or medical response in a tissue, system, animal, or human sought by a researcher, veterinarian, physician, or other clinician who administers the compound-containing composition.

[0263] The “effective dose” will depend on many factors, including the potency of the specific conjugate. When determining the concentration of the compound a patient should receive, the patient’s weight and age may also be factors for those skilled in the art.

[0264] The phrases “administer” and / or “administer compound” should be understood to mean providing a patient in need of treatment with a conjugate or a pharmaceutical composition containing a conjugate as defined herein.

[0265] The recipient of the conjugate as defined in this article can be a person, male or female.

[0266] Optionally, the receptor for the nanoparticles, aggregates, or conjugates; or the pharmaceutical composition comprising nanoparticles, aggregates, or conjugates, may also be a non-human animal. "Non-human animal" refers to the animal kingdom, excluding humans, and includes male or female vertebrates and invertebrates, including: warm-blooded animals, including mammals (including, but not limited to, primates, dogs, cats, cattle, pigs, sheep, goats, rats, guinea pigs, horses, or other cattle, sheep, horses, dogs, cats, rodents, or rat species), birds, insects, reptiles, fish, and amphibians.

[0267] Recipients of conjugates and pharmaceutically acceptable compositions are referred to herein by the interchangeable terms “patient,” “recipient,” “individual,” and “subject.” These four terms are used interchangeably and refer to any person or animal as defined herein (unless otherwise indicated). A patient may receive or has received an endovascular intervention. In one example, the patient is receiving an endovascular intervention. In another example, the patient has received an endovascular intervention. Vascular injury may result from the endovascular intervention. For example, the endovascular intervention may be a stent or balloon angioplasty. In one example, the endovascular intervention may be stent implantation. In another example, the endovascular intervention is balloon angioplasty.

[0268] Diseases, conditions, or illnesses that can be treated with the conjugates described herein include, but are not limited to, vascular diseases such as atherosclerosis, peripheral artery disease, and acute coronary syndrome; gastrointestinal diseases; and kidney diseases.

[0269] Any discussion of documents, bills, materials, devices, articles, etc., included in this specification should not be construed as an admission that any or all of these contents form part of the prior art or are common general knowledge in the field related to this invention, as they existed prior to the priority date of each of the appended claims.

[0270] Example

[0271] Example 1. Surface binding of interleukin to nano-P3

[0272] Carrier-free recombinant rat IL-4 and IL-10 (R&D System, USA) were dissolved in phosphate-buffered saline (PBS) at a stock concentration of 100 ng / μl. To establish binding kinetics, each interleukin was labeled with a Lightning-link Cy5 antibody tag (Novus Biologicals, USA). For each 1 × 10⁻⁶ ppm in ultrapure water (Thermofisher, USA),... 9 Nanoparticles of P3 (200 nm in diameter) were added with 1.42 μg of IL-4 or 1.32 μg of IL-10. Additional water was added to bring the total reaction volume to 1 ml, and the mixture was incubated at room temperature for 1 hour. After 1 hour of incubation, the residue was washed, and the binding kinetics were determined using a Clariostar monochromator microplate reader (BMG Laboratory Technology, Germany).

[0273] The binding efficiency of nano-P3 to IL-4 in solution was the highest at 99%, corresponding to 0.5 ± 0.01 μg / 10 9 Particle loading. The loading capacity was further increased to 1.1 ± 0.02 μg / 10. 9 Particles were observed, but the binding efficiency was low, at 53.1%. The maximum binding efficiency of nano-P3 with IL-10 was 99.9%, representing 0.5 ± 0.02 μg / 10⁻⁶. 9 Total mass loading capacity of the particles. Further loading of IL-10 onto nano-P3 was observed up to 0.80 ± 0.02 μg / 10⁻¹. 9 Particles, corresponding to a binding efficiency of 40%. All experiments were conducted at room temperature in ultrapure water (pH = 6.5). Incubation time was 30 minutes.

[0274] Figure 1 A is a schematic diagram of the functionalization of nano-P3. Small molecules, imaging agents, targeting ligands, or proteins are incubated with nano-P3 to functionalize it.

[0275] Figure 1 B shows the macrophage response to IL-4 and IL-10. IL-4 and IL-10 can transform the phenotype of M1 macrophages (pro-inflammatory) into that of M2 macrophages (anti-inflammatory).

[0276] Figure 2 The loading capacity of IL-4 and IL-10 on nano-P3 was demonstrated. The binding efficiency of IL-4 and IL-10 was 100%. Emission spectroscopy confirmed that nano-P3 could bind to IL-4 and IL-10.

[0277] Example 2. IL-4 bound to nano-P3 induces polarization in M2 macrophages.

[0278] Directing macrophage phenotypes to their M2 anti-inflammatory state has the potential to mitigate further progression of vascular damage and promote disease regression. Various cytokines from the interleukin-1 family, including IL-4 and IL-10, promote the phenotype shift from M1 to M2.

[0279] The inventors sought to investigate the effect of NP3+IL-4 on macrophage polarization in vitro. The study was conducted in 96-well plates at a concentration of 5 × 10⁻⁶. 3 Raw246.7 mouse macrophages (ATCC, USA) were cultured in cells / wells. IL-4-binding nanoparticles of P3 were added at 1 × 10⁻⁶ cells / well. 5 Nanoparticle P3 / pore concentrations were added to macrophage cultures. After 24 hours, macrophages were fixed in 4% paraformaldehyde before scanning electron microscopy (SEM) and confocal imaging. Confocal staining was performed using actin cytoskeleton staining (Abcam, USA) and anti-arginase-1 antibody (Abcam, USA).

[0280] Figure 3 The results showed that nano-P3-IL4 induced M2 activation compared to untreated macrophages and macrophages treated with nano-P3 alone. Confocal staining confirmed that the highly expressed M2 enzyme ARG-1 was significantly upregulated in macrophages treated with nano-P3-IL4, further confirming M2 activation.

[0281] Example 3. Rat carotid artery injury and nano-P3 delivery model (I)

[0282] Using a rat carotid artery model with vascular injury, the efficacy of NP3-binding interleukins in treating cardiovascular pathology was determined in vivo. Figure 1 C).

[0283] Research approval was obtained from the Sydney Local Health District Animal Welfare Committee (Program No. 2017 / 006). Experiments were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purpose. Rats (spreading stage, male, 7 weeks old) were purchased from Laboratory Animal Services (NSW, Australia). Rats were anesthetized by a single intramuscular injection of ketamine (75 mg / kg) and medemidin (0.5 mg / kg). The common carotid artery was dissected, with double ligatures spaced approximately 1 cm apart. A small incision was made distally, through which micro-forceps (World Precision Instruments, USA) were inserted, expanded to their full width, and rotated 360 degrees to damage the entire luminal surface area of ​​the vessel. This was repeated 5 times before the forceps were withdrawn, and a 22G catheter was inserted into the same incision. Through the catheter, a nano-P3 solution bound to IL-4 or IL-10 was administered at a rate of 2 × 10⁻⁶. 8 The concentration of nano-P3 was injected into a total volume of approximately 80 μl of RPMI medium. The nano-P3 solution was incubated for 2 minutes and then completely removed from the container. The incision was sutured with 9-0 nylon sutures, and the two ligatures were loosened to re-establish blood flow. The dissected vessel segment was removed for pathological evaluation after 14 days.

[0284] Figure 4 B shows that once blood flow is restored, free IL-4 is immediately washed away from the vessel wall. However, IL-4 bound to nano-P3 is significantly retained in the container and persists at significant levels after 5 days.

[0285] Figure 5 The formation of new intima was inhibited. Immunostaining (yellow / green) of M2 macrophages in the treated carotid segment showed a significant increase in the NP3+IL-10 group compared to ablation, NP3+IL-4, and free IL-10. Immunostaining assessment of endothelial repair showed that NP3+IL4 and NP3+IL-10 restored complete endothelial integrity 14 days post-injury. However, this was not observed with treatment using free IL-10.

[0286] Fourteen days in vivo, the vascular explants were fixed overnight in 4% PFA, dehydrated with ethanol, and then embedded in paraffin. The embedded vascular segments were then longitudinally segmented into 5 μm thick sections. M2 macrophages and luminal endothelium were stained using anti-CD206 (Abcam, USA) and anti-onWillebrand Factor (Sigma, USA) antibodies. Fluorescence imaging was performed using the Alexa-fluor 594 secondary antibody. Neointima formation was stained using hematoxylin and eosin (H&E) staining. Normal proliferation was calculated as the total area of ​​proliferation divided by the area of ​​the original vascular lumen.

[0287] Analysis of neointimal formation two weeks after therapeutic nano-P3 delivery showed that vascular occlusion was reduced to approximately 35% and 20% of the cross-sectional lumen area in the NP3+IL-4 and NP3+IL10 groups, respectively. Free IL-10 and nano-P3 alone had no significant effect on vascular occlusion, indicating that the nano-P3 platform promotes the therapeutic benefit of IL-10 (Figure 6).

[0288] Example 4. Rat carotid artery injury and nano-P3 delivery model (II)

[0289] Using a rat carotid artery vascular injury model, the efficacy of NP3+IL-10, NP3+sirolimus, or NP3+sulindac in treating cardiovascular diseases was determined in vivo. Vector-free recombinant rat IL-10 (1.34 μg / 10) 9 Nano P3; R&D Systems, USA), Sirolimus (2.50 μg / 10 9 Nano P3; Rapamycin (Sigma-Merck, USA), Sulindac (3.05 μg / 10) 9 Nano P3 (Sigma-Merck, USA), or Cy7 fluorescent tag (5.03 μg / 10⁻⁶). 9 Nano P3; CF750 antibody labeled (Sigma-Merck, USA) was diluted in sterile water and conjugated to 2 × 10⁻⁶. 9 Nano P3.

[0290] Rats suffered vascular injury as described in Example 3, except via catheter, at 2 × 10 8 The concentration of nano-P3 (IL-10, 0.268 μg; sirolimus, 0.5 μg; sulindac, 0.61 μg; Cy7, 1.01 μg) was approximately 80 μl in RPMI medium, and the nano-P3 solution containing IL-10, sirolimus, or sulindac was injected.

[0291] Figure 7The delivery of NP3+IL-10, NP3+sirolimus, or NP3+sulindac showed that, compared with the delivery of the free reagent in a rat carotid artery injury model, the delivery of NP3+IL-10, NP3+sirolimus, or NP3+sulindac led to the inhibition of neointimal hyperplasia.

[0292] The re-endothelialization of blood vessels in a rat carotid artery model treated with NP3+IL-10, NP3+sirolimus, or NP3+sulindac was also investigated using von Willebrand factor (vwf) staining as described in Example 3. Sirolimus is a known agent that impairs wound healing. Surprisingly, the inventors also showed that, although sirolimus has been previously described as impairing wound healing, both NP3+sirolimus and NP3+IL10 stimulated healing (endothelialization). Figure 8 ).

[0293] Example 5. IL-10 and sulindac bound to nano-P3 induce M2 macrophage polarization.

[0294] The inventors sought to investigate the effects of NP3+IL-10 and NP3+sulindac on macrophage polarization in vitro.

[0295] In a 96-well plate, at 5×10 3 Raw246.7 mouse macrophages (ATCC, USA) were cultured in cells / wells. IL-10 (1.34 μg / 10⁻¹) 9 Nano P3) and sulindac (3.05 μg / 10) 9 Nanoparticles (P3) are combined with nanoparticles as described in Example 4.

[0296] NP3+IL-10 or NP3+sulindac at 1×10 5 Nanoparticle P3 / pore concentrations were added to macrophage cultures. After 24 hours, macrophages were fixed in 4% paraformaldehyde before scanning electron microscopy (SEM) and confocal imaging. Confocal staining was performed using actin cytoskeleton staining (Abcam, USA) and anti-arginase-1 antibody (Abcam, USA).

[0297] Figure 9 The results showed that NP3+IL-10 induced M2 activation compared to untreated macrophages (“stripped”) and macrophages treated with nanoparticle P3 alone (“+NP3”). Confocal staining confirmed that the highly expressed M2 enzyme ARG-1 was significantly upregulated in macrophages treated with NP3+IL10, further confirming M2 activation.

[0298] Example 6. Rabbit iliac bone injury and nano-P3 release model

[0299] The efficacy of NP3+IL-10 treatment was evaluated using a rabbit iliac bone injury model.

[0300] The study was approved by the University of Sydney Animal Ethics Committee (AEC) protocol 2019-1653. The experiment was conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. After blunt dissection of the muscle layer, an incision (~1 cm long) was made between the groin skin and the exposed right femoral artery. The artery was ligated distally and above the ligation line, creating a small incision to allow insertion of a 5F sheath (Abbott, TREK coronary dilation catheter, 3.25 mm). The sheath was secured in place with sutures (3-0, silk). A 0.014-inch guidewire was inserted through the sheath and retrogradely into the abdominal aorta. A 3.25 mm angioplasty balloon catheter was then inserted onto the guidewire and advanced into the aorta. Before angiography, NP3 delivery and the location of surrounding vessels were determined by intravenous injection of contrast agent ISOVUE 370 or OMNIPAQUE (75.5g / 100ml), 25%-50% (v / v) contrast agent / saline.

[0301] Iliac artery stripping was performed by inflating a balloon at the bifurcation of the abdominal aorta to a diameter of 3.1 mm and slowly withdrawing it to the femoral artery, repeated three times (1 minute each time). Through the main incision, the same balloon was reinserted into the injured iliac bone at the bifurcation and inflated to 6-8 atm to occlude proximal blood flow. Immediately after blood flow occlusion, IL-10 nanoparticle P3 solution (3×10⁻⁶) was delivered through a sheath. 8 Apply 2 ml of solution to the artery to inflate it and incubate for 2 minutes, then aspirate the remaining solution through the sheath. Remove the sheath, permanently ligate the femoral artery with 3-0 silk sutures, and then remove the sheath. Suture the exposed area with 3-0 silk sutures, using single and double sutures.

[0302] Figure 10 The results show the in vivo performance of IL-10-conjugated 200nm NP in a rabbit iliac artery injury model. A) Rabbit iliac artery injury model; B) H&E staining for neointimal hyperplasia; C) CD31 staining for thrombus formation (white dashed line); D) CD68 staining for inflammatory macrophage infiltration (white staining).

[0303] Vascular treatment with NP3+IL10 showed reduced occlusion, thrombosis, and inflammation.

[0304] Figure 11Compared to the untreated control, NP3+IL-10-treated vessels showed reduced proliferation over seven days. Additionally, the incidence of thrombosis increased over seven days, and this was significantly reduced in NP3+IL-10-treated vessels. CD68 staining for vascular inflammation showed that NP3+IL-10 reduced pro-inflammatory macrophage infiltration and inflammation over seven days compared to the untreated control.

[0305] Example 7. Retention of nano-P3 in a rat carotid artery injury model

[0306] The retention of 200 nm and 100 nm diameter nanoparticles (NP3) was investigated in a rat carotid artery injury model.

[0307] Cy7 fluorophores are conjugated with 200 nm or 100 nm diameter P3 nanoparticles.

[0308] As described in Example 3, rats were subjected to vascular injury, except via catheter, at a rate of 2 × 10 8 The concentration of nano-P3 was determined by injecting a solution containing Cy7 bound to either 100 nm nano-P3 or 200 nm nano-P3 into approximately 80 μl of RPMI medium.

[0309] Figure 12 The results showed that both 100nm and 200nm P3 nanometers were retained after seven days, and 100nm P3 nanometers were also retained two weeks after delivery.

[0310] Nanoparticles of P3 were detected after 14 days when any size was used. The retention curve varied somewhat depending on the size of the nanoparticles of P3 used. Therefore, a particularly preferred retention time can be achieved by selecting an appropriate nanoparticle size of P3.

Claims

1. Use of a conjugate in the preparation of a medicament for treating or preventing neointimal hyperplasia or restenosis, wherein the conjugate comprises: (a) A polymeric nanoparticle with an average diameter of 1 nm to 50 nm formed by plasma, wherein the plasma comprises at least one monomer selected from olefins, alkynes, cycloalkenes, cycloalkynes, or mixtures thereof; or an aggregate comprising two or more of the aforementioned polymeric nanoparticles, wherein the aggregate has an average diameter of 5 nm to 500 nm; and (b) Interleukin-4 or interleukin-10; The conjugate is formulated for delivery to the vascular region of a patient in need.

2. The use according to claim 1, wherein the conjugate is formulated for delivery to the region of the blood vessel using a catheter.

3. The use according to claim 2, wherein the catheter is an occluded perfusion catheter or a effusion balloon catheter.

4. The use according to any one of claims 1 to 3, wherein the neointimal hyperplasia or restenosis occurs as a result of endovascular intervention.

5. The use according to any one of claims 1 to 3, wherein the conjugate is retained in the region of the blood vessel for a longer period than the unconjugated bioactive agent is retained in the region of the blood vessel.

6. The use according to claim 5, wherein the conjugate is retained at the delivery site in the blood vessel for at least 1 day.

7. The use according to claim 6, wherein the conjugate is retained at the delivery site in the blood vessel for at least 5 days.

8. The use according to any one of claims 1 to 3 and 6 to 7, wherein the plasma comprises at least one alkyne.

9. The use according to claim 8, wherein the at least one alkyne is acetylene.

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